Enzyme-resistant compositions containing penicillin in combination with 6-apa

ABSTRACT

This invention has for its objects the provision of compositions of 6-APA with penicillins since it has been found that 6-APA protects penicillins from the action of Beta -lactamases produced by many organisms which destroy many types of penicillins. The 6-APA is provided in a ratio of 1 gram of 6-APA to 20 grams of a given penicillin up to 200 grams of 6-APA to one gram of penicillin. The penicillin is provided in standard dosages for the particular penicillin employed, and in the usual dosage forms. By penicillin is meant that any acyl derivative of 6-aminopenicillanic acid, including salts, esters and amides thereof. By 6-APA is meant 6-aminopenicillanic acid of which the salts, esters and amides thereof may also be employed.

United States Patent [191 Donovick 1 Feb. 18,1975

ENZYME-RESISTANT COMPOSITIONS CONTAINING PENICILLIN IN COMBINATION WITH 6-APA 3,225,033 12/1965 Johnson et al. 424/271 3,245,983 4/1966 Doyle et a1. 424/271 Primary Examiner-Albert T. Meyers Assistant Examiner-Daren M. Stephens Attorney, Agent, or FirmLawrence S. Levinson; Merle J. Smith [57] ABSTRACT This invention has for its objects the provision of compositions of 6-APA with penicillins since it has been found that 6-APA protects penicillins from the action of B-lactamases produced by many organisms which destroy many types of penicillins.

The 6-APA is provided in a ratio of 1 gram of 6-APA to 20 grams of a given penicillin up to 200 grams of 6-APA to one gram of penicillin.

The penicillin is provided in standard dosages for the particular penicillin employed, and in the usual dosage forms. By penicillin is meant that any acyl derivative of 6-amino-penicillanic acid, including salts, esters and.

amides thereof. By 6-APA is meant 6-aminopenicillanic acid of which the salts, esters and amides thereof may also be employed.

10 Claims, No Drawings ENZYME-RESISTANT COMPOSITIONS CONTAINING PENICILLIN IN COMBINATION WITH 6-APA This patent is a continuation of US. Pat. No. 613,406, filed Feb. 2, 1967.

This invention relates to novel therapeutic compositions. More particularly, this invention relates to certain antibiotic compositions comprising a penicillin and 6-aminopenicillanic acid (6-APA) or a salt, ester or amide thereof.

The art has long been aware of the problem of resistance on the part of some microorganisms to treatment with penicillin-containing drugs, in many instances, necessitating the search for other therapeutic agents which are effective against these microorganisms.

Such resistance results from the production of en zymes, such as B-lactamase, by such microorganisms which operate to break down the structure of the penicillin molecule and thus destroy its effectiveness.

It has now been discovered that 6-APA can slow down or interfere with the enzymic breakdown of penicillins, particularly with the action of such enzymes as B-lactamases. Thus, microorganisms which are resistant to penicillins by virtue of their production of such enzymes are rendered more susceptible to the action of the penicillins when 6-APA is included in the formulation.

The compositions of this invention are directed against all types of microorganisms which are naturally resistant or become resistant to penicillins by virtue of their ability to produce enzymes which destroy penicillins, particularly the B-lactamases.

The penicillins included within the scope of this invention are any of those which are susceptible to degradation in the presence of the enzymes known as the B-lactamases. More particularly, the penicillins included within the scope of this invention would encompass acids of the formula wherein X is selected from the group consisting of oxygen and sulfur, and R represents a member selected from the group consisting of aryl, saturated and unsaturated alkyl, aralkyl, alicyclic heterocyclic and substituted derivatives thereof.

More specifically, the aliphatic radicals represented by R include straightchain, branched-chain, saturated and unsaturated radicals, such radicals containing an interrupting group such as oxygen, sulfur, nitrogen, sulfonyl, as a member of the chain, and those containing one or more of the various substituent groups such as halogen, alkoxy, aryloxy, carbonyl, carboxyl, carboxamido, carboalkoxy, carboaryloxy, cyano, sulfo, phosphoro, nitro, amino, alkylamino, arylamino, acrylamino, alkyl, acyloxy, acyl, hydroxyl, mercapto, alkyl mercapto, arylmercapto, as well as aryl, heterocyclic and alicyclic and substituted derivatives thereof along the chain. The carbocyclic radicals. that is, the aryl, aralkyl. polycyclic and monocyclic alicyclic radicals, within the ambit of R include fully saturated and partially or completely unsaturated carbocyclic nuclei and substituted derivatives thereof; e.g., phenyl, naphthyl, phenanthryl, anthryl, styryl, cyclobutyl, Z-methylcyclopentyl, cyclohexyl, a-cyclohexylpropyl, benzyl, naphthylmethyl, decalyl, cyclopentanopolyhydrophenanthryl, and substituted derivatives thereof wherein the substituent or substituents is selected from the group consisting of hydroxy, alkoxy, aryloxy, thiol, thioalkyl, thioaryl, cyano, nitro, carbonyl, acyl, alkyl, aryl, amino, monoand dialkyl amino, monoand diaryl amino, carboxyl, carboxamido, monoand dialkyl carboxamido, monoand diaryl carboxamido, carboalkoxy, carboaryloxy, sulfonic, phosphoric, carboxyhydrazido and azo. The heterocyclic radicals within the scope of R likewise include the fully saturated and partially or completely unsaturated heterocyclic nuclei. that is, monocyclic and polycyclic or condensed heterocyclic radicals having a 5 or 6-membered ring containing at least one of the hetero atoms oxygen, sulfur, nitrogen, and substituted derivatives thereof. Such heterocyclic radicals include, for example, the monocyclic radicals pyrryl, pyridyl, pyrazinyl, pyrrolidyl, piperidyl, pyrimidinyl, oxazolyl, furyl, thienyl, a-pyranyl, y-pyranyl, thiazolyl, imidazoyl; the condensed heterocyclic radicals benzofuryl, thionaphthyl, quinolyl, acridyl, isoquinolyl, indolyl, indazolyl, dihydrobenzofuryl, dibenzofuryl, benzimidazolyl, benzoxazolyl, and substituted derivatives thereof. Also included within the scope of this invention are the nontoxic pharmaceutically acceptable esters, amides and salts of the above compound.

The nontoxic, pharmaceutically acceptable salts include metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted-ammonium salts, e.g., salts of such nontoxic amines as trialkylamines, including triethylamine, procaine, dibenzylamine, N-benzyl-B-phenethylamine, l-ephenamine, N,N'-dibenzylethylenediamine, dehydroalicetylamine, N,N-bis-dehydroalicetylethylenediamine, N-(lower- )alkylpiperidines, e.g., N-ethylpiperidine, and other amines which have been used to form salts with benzylpenicillin. The term (lower)alkyl as used herein means both straight and branched chain aliphatic hydrocarbon radicals having from one to ten carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, amyl, hexyl, 2-ethylhexyl, heptyl, decyl, etc.

The esters and amides included within the scope of the present invention are easily hydrolyzed esters and amides which are converted to the free acid form by chemical or enzymatic hydrolysis.

In those cases wherein the penicillin is amphoteric in nature, for instance, ampicillin, this invention also includes salts of such penicillins formed with nontoxic, pharmaceutically acceptable, organic and inorganic acids. Exemplary of such organic salts are those with maleic, fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic, methylsulfonic, ethanesulfonic, acetic, propionic, tartaric, salicyclic, citric, gluconic, lactic, malic, mandelic, cinnamic, citraconic, aspartic, stearic, plamitic, itaconic, glycolic, p-aminobenzoic. glutamic benzene sulfonic and theophylline acetic acids as well as the S-halotheophyllines, for example, 8-chlor0theophylline and 8-bromotheophylline. Exemplary of such inorganic salts are those with hydrochloric, hydrobromic, sulfuric, sulfamic. phosphoric and nitric acids.

Specific penicillins preferred for use in the compositions of this invention include such as benzylpenicillin, a-phenoxypropylpenicillin, 2,6-dimethoxypenicillin, ot-phenoxymethylpenicillin, a-aminobenzylpenicillin, Z-biphenylpenicillin, 3-phenyl-5- methylisoxazolepenicillin, 3-(4-chlorophenyl)-5- methylisoxazolepenicillin, 2(2,6-dichlorophenyl)-5- methyloxazolepenicillin, 2-ethoxynaphthylpenicillin, and 3-carboxy-2-quinoxalinecarbonylpenicillin. The nontoxic, pharmaceutically acceptable salts, esters and amides of 6-APA included within the scope of the invention are the same as those recited above in regard to the penicillins of this invention. 6-APA, being amphoteric in nature, will also form the acid-addition salts recited above, and hence, these are also included within the scope of this invention.

Particularly preferred compositions of this invention are those comprising Penicillin G or Ampicillin in combination with 6-aminopenicillanic acid.

The compositions of this invention may be administered so as to provide standard dosages for the particular penicillin employed. In general, such dosages contain from 0.01 to 1.5 grams of the penicillin per kilogram of body weight in combination with sufficient 6-APA to protect the penicillin from the action of the enzyme. The proportion of 6-APA to the penicillin should be from 1-20 to 200-1, more particularly 1-12.5 to 160-1. Particularly preferred proportions of 6-APA to the penicillin are from lto 5-1.

To illustrate the effect of B-lactamase on benzylpenicillin and the protective effect of the additional presence of t'i-APA. the following test procedure is followed.

Sufficient hcnzylpenicillin is dissolved in distilled water to give a solution containing 12.5 mg. per ml. To a series of 13 X 100 mm test tubes is added 0.6, 0.4, 0.3 and 0.2 ml. of the penicillin solution. 6-APA is dissolved in 0.4M borate buffer, pH 8.4 to give a solution containing mg./ml. 0.6 MI. of the 6-APA solution is added to each of the above test tubes. To each of these test tubes is then also added 0.1 ml. of a standard commercial penicillinase (B-lactamase) solution containing 50,000 units of enzyme per ml. Finally added to the test tubes is 0.5 ml. of 0.2M phosphate buffer (pH 7.5) and enough water to give a final volume of 2 ml. Thus each test tube contains the following:

remove the residual penicillin. Filter paper discs, inch, are saturated with the n-pentylacetate solutions and then permitted to air dry. The dry discs are then placed on agar plates seeded with Staphylococcus aureus and incubated overnight at 37C. The zones of inhibition are then measured and the residual active penicillin calculated in the usual fashion against a standard curve obtained with penicillin solutions treated as above except that no 6-APA or enzyme was added. The results obtained are shown in Table 1 below, wherein the size of the zone of inhibition is taken as 100% inthe absence of B-lactamase and 6-APA.

TABLE 1 Protection of Penicillin G Residual Active To illustrate the effect of combinations of 6-APA and a-aminobenzylpenicillin on clinically important microorganisms, the following test procedure is employed.

Solutions of the compounds (6-APA and a-aminobenzylpenicillin) are prepared in either trypticase soy broth or penassay broth to give concentrations ranging from 2 to 250 meg/ml. and 0.1 to 25 mcg./ml., respectively. The test procedure for determining the activity of combinations is generally that described by Oswald et al.,' Antibiotic Combinations: An in vitro study'of anti-staphylococcal effects of Erythromycin plus penicillin, Streptomycin, or Tetracycline, Antimicrobial Agents and Chemotherapy (1961 pp. 904-910.

The results of such experiments are set forth in Table 11 below, and indicate the advantages of the instant in- The test tubes are placed in a 30C. water bath for 30 minutes, after which 0.1 ml of 3N H 80, is added to stop all enzymatic action. The contents of the test tubes vention, namely that combinations of 6-APA and ampicillin are more active (as shown by reduced minimal inhibitory concentrations [M.K.C.]) than either comare then extracted with one ml. of n-pentylacetate to ponent alone.

TABLE 11 Effects of Combinations of 6-APA and a-Aminobenzylpenicillin ORGANISM MlC (ug./ml.)

Combination* Microbial Species Strain 6-APA Ampicillin o-APA Ampicillin Proteus vulgaris 3855 63 0.4 4 0.1 Proteus mirabilis 3873 63 0.8 16 0.1 Salmonella lyphi- 3821 31 0.4 4 0.1

murium Salmonella galli- 3030 16 0.8 4 0.1

narum Shigella flexneri 1461 63 0.8 8 0.1 Esl'heridua coll 3471 16 0.8 4 0.1 Exrlteriduu coli 2975 31 3.1 2 1.6 Aerubut'rer uemgenes 3425 3| 1.6 8 0.2 At'ruhar'lcr aamgenes 167K 3 1 1 3. l Aemhm'tcr species 3537 4 6.3 2 0.2 Arm/Javier species 3536 31 25 l 3 Arm/mater species 3534 63 25 I 6.3 lseudrmmnas species 3547 31 3.1 16 0.8 lseudnmrmas species 3550 250 31 63 2 Pseudomonas aerugi- 5943 250 50 2 25 HOS Klebriella pneumoniae 1565 125 25 16 6.3

End point employing minimum concentration of ampicillin.

The compositions provided by the present invention comprise mixtures of a penicillin as provided above in admixture with 6-APA. If desired, this combination may be incorporated with a pharmaceutically acceptable carrier. Further, other therapeutic agents, such as other antibiotics may be included in the compositions of the present invention, for instance, sulfonamides, streptomycin, amphotericin B, nystatin, etc. The compositions may take any of the well-known medicinal dosage forms, e.g., tablets, lozenges, solutions or suspensions.

The following examples illustrate the preparation of various formulations which are encompassed within the scope of the instant invention. The formulations exemplified. however, are not intended to be limitative of the invention since other obvious modifications will occur to one skilled in the art, to which this invention also applies equally.

Example 1 Sodium Ampicillin. sterile fi-APA. sterile man Aseptically blend the ingredients to provide a dry powder for reconstitution. Fill into sterile vials and seal.

Example 2 Penicillin G Potassium. sterile 6-APA. sterile Aseptically blend the powders. Fill into sterile vials at a level of l gm./vial of Penicillin G activity.

Aseptically dissolve the ingredients and add the sterile procaine penicillin G. Stir to homogeneity. Aseptically fill the sterile suspension into vials and seal.

Example 4 Ampicillin trihydrate (sufficient to supply 230.2 gms. mg. of ampicillin per 5.0 cc. plus a 10% excess) 6-APA 46.0 gms. Saccharin. U.S.P. 7.2 gms. Sodium Cyclamate 21.6 gms. Sodium Citrate gms. Sodium Carboxymethylcellulose.

low viscosity 7.0 gms. FD&C Red No. 2 1.5 gms. Mixed Fruit Flavor. dry 27.0 grns.

Sugar. fine powder. enough to make Make a homogeneous mix of all the powders, sift through a 20 mesh screen and reblend. The powder mixture is filled into glass bottles at the rate of 40 gm. per bottle (sufficient for 12 doses). The suspension is prepared by the pharmacist at the time of'dispensing by adding 35 cc. of distilled water, to make a final volume of 60 cc., and shaking. The suspension so prepared is stable for at least seven days when stored in a refrigerator (about 5C).

Make a homogeneous mix of all the powders, sift through a 20 mesh screen and reblend. The mixture is filled into amber bottles at the rate 80 gm. per bottle 7 (sufficient for 30 doses). The suspension is prepared by the pharmacist at the time of dispersing by adding 105 cc. of distilled water to make a total volume of 150 cc., and shaking.

What is claimed is:

l. A method of protecting penicillin from the action of microorganisms which produce B-lactamase enzymes which destroy penicillin which comprises admin- EXamPle 6 istering a standard dosage form of the penicillin together with an amount of free 6-aminopenicillanic acid Penicillin G Potassium (to provide 200,000 units 125.3 mg. or pharmaceutically acceptable salts esters and amides of penicillin) ca. thereof, the amount being effective to prevent destrucg'm 5288 :5: tion of the penicillin by B-lactamase enzymes, the Polyvinylpyrrolidone 45.4 mg. amount of penicillin in the dosage form being from mg swarms about 0.01 to about 1.5 grams per kilogram of body Corn Starch 25.0 mg weight. f spray D'icd 9504) mg 2. A method according to claim 1 wherein the dosage form is a tablet.

3. A method according to claim 1 wherein the dosage Make a homogeneous mix of the penicillin G, 6- fOrm is a loze ge. APA, chalk, polyvinylpyrrolidone, and corn starch and 4. A method according to claim 1 wherein the dosage one half of the magnesium stearate and talc. Slug on a form is a solution. rotary tablet press and reduce the slugs to form a gran- 5. A method according to claim 1 wherein the dosage ulation. Add the remainder of the talk and magnesium form is a suspension. stearate, and sufficient lactose spray dried U.S.P. to 6. A composition comprising a standard dosage form make the batch weight. Compress on a suitable rotary of penicillin together with an amount of free 6 tablet press. aminopenicillanic acid, or pharmaceutically acceptable Example 7 Ampicillin trihydrate (to provide 250 mg. 5% excess) ca. 300.0 mg. 6-APA ca. 75.0 mg. Magnesium Slearate 9.0 mg Milk Sugar U.S.P. q.s. to (ca. ll6.0 mg.) 500.0 mg

Make a homogeneous mix of the powders and slug on salts, esters or amides thereof wherein the esters or ama rotary tablet press. Reduce the slugs to fine powder ides are formed by reaction of the carbo xyl group of and fill the material into No. 0 empty capsules on a the acid, the amount of 6-ammopenicillanic acid n the semi-automatic or automatic capsule filler. composition being effective to prevent destruction of the penicillin by B-lactamase enzymes, the amount of v Example 8 -aminopenicillanic acid to penicillin in the dosage fomi being from about l-20 to 200-]. p i Penicillin Smile 3000 7. A standard dosage form of penicillin according to PA. sterile I 7.5 gclaim 6 wherein the-dosage form is a tablet.

'g' z gg ff' t A standard dosage form of penicillin according to 70M. sterile 2.0 mg. claim 6 wherein the dosage form is a lozenge;

9. A standard dosage form of penicillin according to Y claim 6 wherein the dosage form is a solution. Asepticany blend the PQWders and into a one'dose 10. A standard dosage form of penicillin according to via] for reconstitution. Multiple doses may also be filled claim 6 wherein the dosage form is a suspension into sterile vials. 

1. A METHOD OF PROTECTING PENICILLIN FROM THE ACTION OF MICROORGANISMS WHICH PRODUCE b-LACTAMASE ENZYMES WHICH DESTROY PENICILLIN WHICH COMPRISES ADMINISTERING A STANDARD DOSAGE FROM THE PENICILLIN TOOGETHER WITH AN AMOUNT OF FREE 6-AMINOPENICILLANIC ACID OR PHARMACEUTICALLY ACCEPTABLE SALTS, ESTERS AND AMIDES THEREOF, THE AMOUNT BEING EFFECTIVE TO PREVENT DESTRUCTION OF THE PENICILLIN BY B-LACTAMASE ENZYMES, THE AMOUNT OF PENICILLIN IN THE DOSAGE FORM BEING FROM ABOUT 0.01 TO ABOUT 1.5 GRAMS PER KILOGRAM OF BODY WEIGHT.
 2. A method according to claim 1 wherein the dosage form is a tablet.
 3. A method according to claim 1 wherein the dosage form is a lozenge.
 4. A method according to claim 1 wherein the dosage form is a solution.
 5. A method according to claim 1 wherein the dosage form is a suspension.
 6. A composition comprising a standard dosage form of penicillin together with an amount of free 6-aminopenicillanic acid, or pharmaceutically acceptable salts, esters or amides thereof wherein the esters or amides are formed by reaction of the carboxyl group of the acid, the amount of 6-aminopenicillanic acid in the composition being effective to prevent destruction of the penicillin by Beta -lactamase enzymes, the amount of 6-aminopenicillanic acid to penicillin in the dosage form being from about 1-20 to 200-1.
 7. A standard dosage form of penicillin according to claim 6 wherein the dosage form is a tablet.
 8. A standard dosage form of penicillin according to claim 6 wherein the dosage form is a lozenge.
 9. A standard dosage form of penicillin according to claim 6 wherein the dosage form is a solution.
 10. A standard dosage form of penicillin according to claim 6 wherein the dosage form is a suspension. 